| This thesis has focused on the procedure of cryoprotectant removal and performed related research in several aspect. First, several key problems of cryopreservation are sketched to give a whole picture. The common methods and materials of cryopreservation and the molecular biological mechanisms of cryoinjury are introduced. Some common cryoprotectants and their usages are also provided.In this study, a theoretical method is presented to optimize the diluent flow rate in the dilution-filtration system to further reduce the osmotic damage to cells and shorten the washing time necessary to remove cryoprotectants, based on a discrete mass transfer concept. In the method, the diluent flow rate is automatically adjusted by a program code in each cycle to maximize the clearance of CPAs, whereas the volume of RBCs is always maintained below the upper volume tolerance limit. The results show that the optimized diluent flow rate can significantly decrease the washing time of CPAs. The washing time under the optimized diluent flow rate can be reduced by over50%, compared to the one under the fixed diluent flow rate. In addition, the advantage of our method becomes more significant when the blood flow rate is lower, the dilution region volume is larger, the initial CPA concentration is higher, or the cell-swelling limit set by the system is smaller. The proposed method for the dilution-filtration system is an ideal solution for not only guaranteeing the volume safety of RBCs but also shortening the washing time of CPAs. In practice, the optimization strategies provided here will be useful in the rapid preparation of cryopreserved blood for clinical use.In the process of removing cryoprotectants, the theoretically optimal operating condition, which is based on the assumption that the distribution of cells is uniform, is often used to reduce or even avoid the hypotonic damage to cells. However, due to the polydispersity of cells, the optimal condition is actually not reliable. In this study, based on the discrete concept developed in our previous work, the effect of the polydispersity on the recovery rate of cells in the dilution-filtration system was statistically investigated by assigning three random parameters, isotonic cell volume, cell surface area, and osmotically inactive cell volume, to cells in small units of blood. The results show that, due to the polydispersity, the real recovery rate deviates from the ideal value that is based on uniform distribution. The deviation significantly increases with the standard errors of cell parameters, and it can be also magnified by high cryoprotectant concentrations. Under the effect of polydispersity, the uniform distribution-based optimized blood or diluent low rate is not perfect. In practice, one should adopt a more conservative blood or diluent flow rate so that the hypotonic damage to cells can be further reduced.Novel methods of cryoprotectant removal based on microfluidics are also investigated in this study. A microfluidic chip for continuous cryoprotectant removal is designed and fabricated, and experiments to evaluate the efficiency of the chip are also performed. The results show that this kind of microfluidic chip can realize the cryoprotectant removal of certain amount of cells. Due to the limit of the flow rate in microfluidics, this method is appropriate for small and precious samples. The factors influencing the efficiency and recovery rate are analyzed, which can have certain reference value to cryoprotectant removal chips or other kinds of mixture-separation structure chips. |
